1. Technical Field
The subject invention relates to methods of producing non-human transgenic mammals which produce various oligosaccharides in their milk. Additionally, the subject invention relates to the generated mammals, the milk which these mammals produce, compositions comprising the milk, fractions of the milk, and the purified oligosaccharides, as well as glycoproteins, present in the milk.
2. Background Information
In the past, several individuals have achieved transgenic expression of primary gene products (i.e., proteins) in the milk of transgenic mammals (U.S. Pat. No. 5,322,775, Wall et al., Proc. Natl. Acad. Sci. USA 88:1696-1700 (1991) and Krimpenfort, P., Cancer Detection and Prevention 17(2):301-305 (1993)). Such expression was carried out in order to produce large quantities of the heterologous proteins in milk (Velander et al., Scientific American, January 1997, pp.70-74).
Recently, it was also demonstrated that expression of secondary transgenic products, for example, oligosaccharides, glycoproteins and glycolipids is, in fact, possible (Prieto et al., Journal of Biological Chemistry 49:29515-29519 (1995)). The experiments carried out by Prieto et al. advanced the concept of using the mammary glands of transgenic mammals as bioreactors and not solely as an alternative protein synthesis system.
Most of the experiments referenced in the above articles were carried out by expression of heterologous transgenes. One reason for such expression is because homologous genes, which are transgenically expressed, may cause difficulties, due to conflicts with internal protein synthesis of mammary gland development (Burdon et al., Mechanisms of Development 36:64-67 (1991)). Another reason why heterologous transgenes are utilized is that most of the experiments which have been carried out are designed for the production of human biological products.
The synthesis of secondary gene products, such as milk oligosaccharides, depends primarily on the expression of glycosyltransferases during lactation. Additionally, there are many benefits to the production of such oligosaccharides. For example, in a transgenic system for the synthesis of oligosaccharides, only catalytic amounts of glycosyltransferases are necessary. Also, the origin of the transgene may not be relevant as long as the secondary gene product or products are the desired one or ones.
It must also be noted that non-human mammals have a variety of glycosyltransferases which have counterparts in the human enzyme repertoire (Thurin et al., The Journal of Biological Chemistry 265(12):7055-7061 (1990)). Some of these transferases have been cloned from the tissues of non-human mammals, for example, mice (Larsen et al., Proc. Natl. Acad. Sci. USA 86:8227-8231 (1989)). Thus, transgenic expression of homologous glycosyltransferases in the mammary glands of non-human mammals represents a means of producing oligosaccharides, in addition to oligosaccharides produced using heterologous glycosyltransferases. Expression of both such enzyme types, as well as the oligosaccharides which are created by the action of such enzymes, and glycoproteins related thereto, are considered to fall within the scope of the present invention.
The oligosaccharides described above may be added, for example, to infant formula so that the formula-fed infant may receive the same benefits as the breast fed infant. For example, it is possible that the oligosaccharides may provide resistance to infection by pathogenic bacteria (Prieto et al., supra). Additionally, such oligosaccharides may be added to other compositions such as nutritional supplements or to pharmaceuticals. The aim of the present invention is to produce non-human transgenic mammals which produce such oligosaccharides, using either homologous or heterologous enzymes including, for example, glycosyltransferases. Such oligosaccharides may then be added to various compositions due to their beneficial properties.
Additionally, the presence of oligosaccharides and other glycoconjugates in the milk of transgenic animals may result in the acquisition of protection or resistance to viral, bacterial or fungal infections or toxins, by the offspring of such animals.